Carbapenem-resistant Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa are emerging multidrug-resistant Gram-negative bacterial pathogens. With increasing frequency, they often prove untreatable or treatable only with toxic antimicrobials. Therefore, the CDC now categorizes such organisms in their top antibiotic resistance threat level. New anti-infective strategies are urgently needed. Fusidic acid is a steroid antibiotic that has activity against S. aureus inclusive of methicillin-resistant strains, M. tuberculosis, and N. gonorrhoeae. It also has an excellent safety profile in humans and can be given by both by oral and intravenous routes. However, it does not penetrate the outer membrane of Gram-negative ESKAPE pathogens, and, therefore, these pathogens are resistant to achievable drug levels. However, experiments performed in combination with colistin and other membrane-permeabilizing agents indicate that even the most highly resistant carbapenemase-producing, colistin-resistant Enterobacteriaceae (expressing NDM-1, KPC, VIM, and/or MCR-1) and also A. baumannii and P. aeruginosa are susceptible to this antibiotic, if outer membrane penetration is facilitated. Further, the major circulating resistance elements in S. aureus that bind to and block the fusidic acid target (EF-Tu) are ineffective against Gram-negative pathogens. Further, target modification- based resistance confers a significant fitness cost. Therefore, pre-existing resistance in Gram-negatives is predicted to be negligible, and non-transferable from Gram-positive organisms. Recently so-called eNTRy rules have defined molecular properties that enhance penetrance into Gram-negatives. Based on intrinsically compelling properties of fusidic acid, we propose to use novel retrosynthetic semi-synthesis approaches to apply eNTRy rules during development of fusidic acid analogues, via amine and fluorine substitution. The underlying goal will be to identify analogues that have broad-spectrum activity against both Gram-positive and Gram-negative pathogens and fill a critical medical need. There are no previously published efforts along these lines, and, therefore, the proposed work is both novel and innovative. Importantly, the analogues will be profiled in a series of complementary functional studies. These will include both in vitro and in vivo measurements that will distinguish effects of penetrance and activity; activity spectrum studies; and assessment of metabolic stability; resistance propensity, and toxicology that will help rank compounds for further intensive exploration. The near-term goal of this two-year, exploratory R21 proposal is to address potential of retrosynthetic application of eNTRy rules to identify tractable, potent, broad-spectrum fusidic acid analogues worthy of further exploration.